Production cell line enhancers

ABSTRACT

The present invention relates to discovery of the ectopic expression of EDEM2 in a production cell to improve the yield of a useful multi-subunit protein. Thus, the present invention provides for production cell lines, such as the canonical mammalian biopharmaceutical production cell, the CHO cell, containing recombinant polynucleotides encoding EDEM2. Also disclosed is a production cell containing both an EDEM2-encoding polynucleotide as well an XBP1-encoding polynucleotide. Improved titers of antibodies produced by these cell lines are disclosed, as well as the improved cell densities attained by these cells in culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is (1) a continuation of U.S. patent application Ser.No. 15/201,104 filed 1 Jul. 2016, now U.S. Pat. No. 9,688,751, and whichis a divisional of U.S. patent application Ser. No. 14/555,220 filed 26Nov. 2014, now U.S. Pat. No. 9,382,315, and which is divisional of U.S.patent application Ser. No. 13/904,587 filed 29 May 2013, now U.S. Pat.No. 9,079,954, and which is a continuation-in-part of PCT InternationalApplication No. PCT/US2013/043116, filed 29 May 2013, which claims thebenefit of U.S. patent application Ser. No. 13/904,587, filed 29 May2013, now U.S. Pat. No. 9,079,954, and claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/652,549filed 29 May 2012; and (2) a continuation of U.S. patent applicationSer. No. 14/737,090, filed on Jun. 11, 2015, which is a continuation ofU.S. patent application Ser. No. 13/904,587, filed on May 29, 2013, nowU.S. Pat. No. 9,079,954, and which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/652,549 filed 29May 2012, each of which is herein specifically incorporated by referencein its entirety.

SEQUENCE LISTING

This application incorporates by reference the sequence listingsubmitted in computer readable form as file 8150US08_ST25.txt created onMay 25, 2017 (206,456 bytes).

FIELD OF THE INVENTION

The invention relates to a cell or cells expressing a recombinantstress-response lectin for the improved production of a multi-subunitprotein. Specifically, the invention provides a mammalian cell andcell-line derived therefrom containing a gene encoding EDEM2, and whichyields antibody at a high titer.

BACKGROUND OF THE INVENTION

The manufacture of therapeutically active proteins requires properfolding and processing prior to secretion. Proper folding isparticularly relevant for proteins, such as antibodies, which consist ofmultiple subunits that must be properly assembled before secretion.Eukaryotic cells have adapted a system that ensures the proper foldingof proteins and the removal of misfolded proteins from the secretorypathway. This system is called the unfolded protein response (UPR)pathway, and it is triggered by the accumulation of misfolded proteinsin the endoplasmic reticulum (ER).

An early event of the UPR is the activation of the transcription factorXbp1, which in turn activates the transcription of endoplasmic reticulumdegradation-enhancing alpha-mannosidase-like protein 2 (EDEM2), a memberof the endoplasmic reticulum associated degradation (ERAD) pathway.EDEM2 facilitates the removal of misfolded proteins. The ERAD pathwaycomprises five steps: (1) chaperone-mediated recognition of malformedproteins, (2) targeting of malformed proteins to the retrotranslocationmachinery or E3-ligases, which involves EDEM2, (3) initiation ofretrotranslocation; (4) ubiquitylation and further retrotranslocation;and (5) proteosome targeting and degradation.

Antibodies are multi-subunit proteins comprising two heavy chains andtwo light chains, which must be properly folded and associated to form afunctional heterotetramer. Any improvement in the efficient and accurateprocessing of the heavy and light chains to improve the yield or titerof functional antibody heterotetramers is desired.

SUMMARY OF THE INVENTION

Applicants made the surprising discovery that the ectopic expression ofEDEM2 in a protein-manufacturing cell line increases the average outputof protein per cell, increases the titer of protein secreted into themedia, and increases the integrated cell density of production celllines.

Thus, in one aspect, the invention provides a cell containing (a) arecombinant polynucleotide that encodes a stress-induced mannose-bindinglectin and (b) a polynucleotide that encodes a multi-subunit protein. Insome embodiments, the stress-induced mannose-binding lectin is an EDEM2protein, non-limiting examples of which are provided in Table 1, and themulti-subunit protein is an antibody. In other embodiments, the cellalso contains a polynucleotide that encodes the active spliced form ofXBP1, non-limiting examples of which are provided in Table 2. In oneembodiment, the cell is a mammalian cell, such as a CHO cell used in themanufacture of biopharmaceuticals.

In another aspect, the invention provides a cell line derived from thecell described in the previous aspect. By “derived from”, what is meantis a population of cells clonally descended from an individual cell andhaving some select qualities, such as the ability to produce activeprotein at a given titer, or the ability to proliferate to a particulardensity. In some embodiments, the cell line, which is derived from acell harboring the recombinant polynucleotide encoding a stress-inducedmannose-binding lectin and a polynucleotide encoding a multi-subunitprotein, is capable of producing the multi-subunit protein at a titer ofat least 3 grams per liter of media (g/L), at least 5 g/L, or at least 8g/L. In some embodiments, the cell line can attain an integrated celldensity (ICD) that is at least 30% greater, at least 50% greater, atleast 60% greater, or at least 90% greater than the integrated celldensity attainable by a cell line derived from what is essentially thesame cell but without the recombinant polynucleotide encoding thestress-induced mannose-binding lectin.

In another aspect, the invention provides an isolated or recombinantpolynucleotide comprising a nucleic acid sequence encoding an EDEM2protein, which is operably linked (cis) to a constitutive andubiquitously expressed mammalian promoter, such as the ubiquitin Cpromoter. In some embodiments, the EDEM2 protein has the amino acid ofSEQ ID NO: 8, or an amino acid sequence that is at least 92% identicalto any one of SEQ ID NO: 1-7. In some embodiments, the polynucleotidecomprises a nucleic acid sequence of SEQ ID NO: 16. In one particularembodiment, the polynucleotide consists of a nucleic acid sequence ofSEQ ID NO: 14; and in another particular embodiment, SEQ ID NO: 15.

In another aspect, the invention provides an isolated or recombinantpolynucleotide comprising a nucleic acid sequence encoding an XBP1protein, which is operably linked to (in cis) a constitutive andubiquitously expressed mammalian promoter, such as the ubiquitin Cpromoter. In some embodiments, the XBP1 protein has the amino acid ofSEQ ID NO: 13, or an amino acid sequence that is at least 86% identicalto any one of SEQ ID NO: 9-12. In some embodiments, the polynucleotidecomprises a nucleic acid sequence of SEQ ID NO: 18. In one particularembodiment, the polynucleotide consists of a nucleic acid sequence ofSEQ ID NO: 17.

In another aspect, the invention provides a cell that contains anEDEM2-encoding polynucleotide, as described in the prior aspect, and apolynucleotide that encodes a multi-subunit protein, such as anantibody. In some embodiments, the cell also contains an XBP1-encodingpolynucleotide, as described in the preceding aspect. In one embodiment,the multi-subunit protein is an antibody, and the heavy chain of theantibody comprises an amino acid sequence of SEQ ID NO: 43 and SEQ IDNO: 44, and the light chain of the antibody comprises an amino acidsequence of SEQ ID NO: 45 and SEQ ID NO: 46. In this and severalembodiments, each polypeptide subunit of the multi-subunit protein isencoded by a separate polynucleotide. Thus, for example, apolynucleotide encoding an antibody may include a polynucleotideencoding a heavy chain and a polynucleotide encoding a light chain,hence two subunits. In some embodiments, the cell is a Chinese hamsterovary (CHO) cell.

In one embodiment, the encoded multi-subunit protein is an anti-GDF8antibody having a heavy chain variable region amino acid sequence of SEQID NO: 20 and a light chain variable region amino acid sequence of SEQID NO: 22. In one embodiment, the anti-GDF8 antibody comprises a heavychain having an amino acid sequence of SEQ ID NO: 19 and a light chainhaving an amino acid sequence of SEQ ID NO: 21. In one embodiment, thepolynucleotide that encodes the heavy chain of the anti-GDF8 antibodycomprises a nucleic acid sequence of SEQ ID NO: 23; and thepolynucleotide that encodes the light chain of the anti-GDF8 antibodycomprises a nucleic acid sequence of SEQ ID NO: 25. In one embodiment,the polynucleotide that encodes the heavy chain of the anti-GDF8antibody consists of a nucleic acid sequence of SEQ ID NO: 24; and thepolynucleotide that encodes the light chain of the anti-GDF8 antibodyconsists of a nucleic acid sequence of SEQ ID NO: 26.

In another embodiment, the encoded multi-subunit protein is an anti-ANG2antibody having a heavy chain variable region amino acid sequence of SEQID NO: 28 and a light chain variable region amino acid sequence of SEQID NO: 30. In one embodiment, the anti-ANG2 antibody comprises a heavychain having an amino acid sequence of SEQ ID NO: 27 and a light chainhaving an amino acid sequence of SEQ ID NO: 29. In one embodiment, thepolynucleotide that encodes the heavy chain of the anti-ANG2 antibodycomprises a nucleic acid sequence of SEQ ID NO: 31; and thepolynucleotide that encodes the light chain of the anti-ANG2 antibodycomprises a nucleic acid sequence of SEQ ID NO: 33. In one embodiment,the polynucleotide that encodes the heavy chain of the anti-ANG2antibody consists of a nucleic acid sequence of SEQ ID NO: 32; and thepolynucleotide that encodes the light chain of the anti-ANG2 antibodyconsists of a nucleic acid sequence of SEQ ID NO: 34.

In another embodiment, the encoded multi-subunit protein is ananti-ANGPTL4 antibody having a heavy chain variable region amino acidsequence of SEQ ID NO: 36 and a light chain variable region amino acidsequence of SEQ ID NO: 38. In one embodiment, the anti-ANGPTL4 antibodycomprises a heavy chain having an amino acid sequence of SEQ ID NO: 35and a light chain having an amino acid sequence of SEQ ID NO: 37. In oneembodiment, the polynucleotide that encodes the heavy chain of theanti-ANGPTL4 antibody comprises a nucleic acid sequence of SEQ ID NO:39; and the polynucleotide that encodes the light chain of theanti-ANGPTL4 antibody comprises a nucleic acid sequence of SEQ ID NO:41. In one embodiment, the polynucleotide that encodes the heavy chainof the anti-ANGPTL4 antibody consists of a nucleic acid sequence of SEQID NO: 40; and the polynucleotide that encodes the light chain of theanti-ANGPTL4 antibody consists of a nucleic acid sequence of SEQ ID NO:42.

In another aspect, the invention provides a method of manufacturing amulti-subunit protein, by culturing a cell of the previous aspect in amedium, wherein the multi-subunit protein is synthesized in the cell andsubsequently secreted into the medium. In some embodiments, themulti-subunit protein is an antibody, such as for example anti-GDF8,anti-ANG2, anti-ANGPTL4, or an antibody having a heavy chain sequence ofSEQ ID NO: 43 and 44, and a light chain sequence of SEQ ID NO: 45 and46. In some embodiments, the multi-subunit protein attains a titer of atleast 3 g/L, at least 5 g/L, at least 6 g/L, or at least 8 g/L. In someembodiments, the cell proliferates in the medium and establishes anintegrated cell density of about .gtoreq.5×10⁷ cell-day/mL, about.gtoreq.1×10⁸ cell-day/mL, or about .gtoreq.1.5×10⁸ cell-day/mL.

In another aspect, the invention provides a multi-subunit protein, whichis manufactured according to the method described in the precedingaspect. In one embodiment, the manufactured protein is an antibody. Insome embodiments, the antibody consists of a heavy chain, whichcomprises an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44, anda light chain, which comprises an amino acid sequence of SEQ ID NO: 45and SEQ ID NO: 46. In one specific embodiment, the manufacturedmulti-subunit protein is an anti-GDF8 antibody having a heavy chainvariable region amino acid sequence of SEQ ID NO: 20 and a light chainvariable region amino acid sequence of SEQ ID NO: 22. In anotherspecific embodiment, the manufactured multi-subunit protein is ananti-ANG2 antibody having a heavy chain variable region amino acidsequence of SEQ ID NO: 28 and a light chain variable region amino acidsequence of SEQ ID NO: 30. In yet another specific embodiment, themanufactured multi-subunit protein is an anti-ANGPTL4 antibody having aheavy chain variable region amino acid sequence of SEQ ID NO: 36 and alight chain variable region amino acid sequence of SEQ ID NO: 38.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows consistently higher protein titer (in mg/L) forEDEM2-expressing clonal cell-lines (gray circles) compared toXBP1-expressing clonal cell-lines (black circles).

FIG. 1B depicts the integrated cell density (cell-days/mL) forEDEM2-expressing clones (gray circles) compared to XBP1-expressingclones (black circles). Each clone (#1-24) expresses the same gene ofinterest (antibody), under the same regulatory conditions, andexpressing either XBP1 (RGC91) or EDEM2 (RGC92) at a transcriptionallyactive locus.

FIG. 2A shows the FACS scans (flow cytometry-based autologous secretiontrap (FASTR)) of several clones expressing XBP1. Inconsistency in thepeaks is indicative of unstable growth (i.e. a variable or heterogeneousmixture) of the cells in the clones tested.

FIG. 2B shows the FACS scans of several clones expressing EDEM2, havinglittle to no variation in the peaks representative of clonal stabilityin the clones tested. FIG. 2B depicts the clonal stability of severalclonal cell-lines expressing EDEM2 and an antibody of interest.

DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about”, when used in reference to a particular recited numerical valueor range of values, means that the value may vary from the recited valueby no more than 1%. For example, as used herein, the expression “about100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2,99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

As used herein, the term “recombinant polynucleotide”, which is usedinterchangeably with “isolated polynucleotide”, means a nucleic acidpolymer such as a ribonucleic acid or a deoxyribonucleic acid, eithersingle stranded or double stranded, originating by genetic engineeringmanipulations. A recombinant polynucleotide may be a circular plasmid ora linear construct existing in vitro or within a cell as an episome. Arecombinant polynucleotide may be a construct that is integrated withina larger polynucleotide molecule or supermolecular structure, such as alinear or circular chromosome. The larger polynucleotide molecule orsupermolecular structure may be within a cell or within the nucleus of acell. Thus, a recombinant polynucleotide may be integrated within achromosome of a cell.

As used herein, the term “stress-induced mannose-binding lectin” refersto a mannose-binding protein, which means a protein that binds or iscapable of binding mannose, derivatives of mannose, such asmannose-6-phosphate, or a glycoprotein that expresses mannose or amannose derivative in its glycocalyx; and whose activity is upregulatedduring stress. Cellular stress includes inter alia starvation, DNAdamage, hypoxia, poisoning, shear stress and other mechanical stresses,tumor stress, and the accumulation of misfolded proteins in theendoplasmic reticulum. Exemplary stress-induced mannose-binding lectinsinclude the EDEM proteins EDEM1, EDEM2 and EDEM3, Yos 9, OS9, and XTP3-B(see Vembar and Brodsky, Nat. Rev. Mol. Cell. Biol. 9(12): 944-957,2008, and references cited therein).

As used herein, the term “EDEM2” means any ortholog, homolog, orconservatively substituted variant of endoplasmic reticulumdegradation-enhancing alpha-mannosidase-like protein. EDEM2 proteins aregenerally known in the art to be involved endoplasmicreticulum-associated degradation (ERAD), being up-regulated by Xbp-1 andfacilitating the extraction of misfolded glycoproteins from the calnexincycle for removal. (See Mast et al., Glycobiology 15(4): 421-436, 2004;Olivari and Molinari, FEBS Lett. 581: 3658-3664, 2007; Olivari et al.,J. Biol. Chem. 280(4): 2424-2428, 2005; and Vembar and Brodsky 2008,which are herein incorporated by reference.) Exemplary EDEM2 sequencesare depicted in Table 1, which is cross-referenced to the SequenceListing.

TABLE 1 % id Animal SEQ ID NO: human % id mouse % id hamster Mouse 1 93100 96 Rat 2 94 98 96 Hamster 3 93 96 100 Human 4 100 93 93 Chimpanzee 599 94 93 Orangutan 6 97 92 92 Zebra fish 7 69 70 69 Consensus 8 100 100100

As used herein, the term “Xbp1”, also known as XBP1 or X-box bindingprotein 1, means any ortholog, homolog, or conservatively substitutedvariant of Xbp1. Xbp1 is a transcription factor and functional elementof the UPR. ER stress activates both (1) the transcription factor ATF6,which in turn upregulates the transcription of Xbp1 mRNA, and (2) the ERmembrane protein IRE1, which mediates the splicing of the precursor Xbp1mRNA to produce active Xbp1. As mentioned above, activated Xbp1 in turnupregulates the activity of EDEM2. (See Yoshida et al., Cell Structureand Function 31(2): 117-125, 2006; and Olivari, 2005.) Exemplary Xbp1amino acid sequences are depicted in Table 2, which is cross-referencedto the Sequence Listing.

TABLE 2 Animal SEQ ID NO % id human % id mouse % id hamster Mouse 9 86100 92 Hamster 10 86 92 100 Human 11 100 86 86 Zebra fish 12 47 47 48Consensus 13 100 100 100

As used herein, the term “antibody” is generally intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM); however, immunoglobulinmolecules consisting of only heavy chains (i.e., lacking light chains)are also encompassed within the definition of the term “antibody”. Eachheavy chain comprises a heavy chain variable region (abbreviated hereinas HCVR or VH) and a heavy chain constant region. The heavy chainconstant region comprises three domains, CH1, CH2 and CH3. Each lightchain comprises a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion comprises one domain (CL1). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementarydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. An “isolated antibody” or “purified antibody” may besubstantially free of other cellular material or chemicals.

The term “specifically binds”, or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of at least about 1×10⁻⁶ M orgreater. Methods for determining whether two molecules specifically bindare well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds human GDF8 (for example) may, however, havecross-reactivity to other antigens, such as GDF8 molecules from otherspecies (orthologs).

Various antibodies are used as examples of multi-subunit proteinssecreted by cells harboring the polynucleotide encoding a stress-inducedmannose-binding lectin. Those examples include anti-GDF8, anti-ANG2, andanti-ANGPTL4 antibodies. These and similar antibodies are described inUS Pat. Apps. No. 20110293630, 20110027286, and 20110159015respectively, which are incorporated herein by reference.

As used herein, the term “cell” refers to a prokaryotic or eukaryoticcell capable of replicating DNA, transcribing RNA, translatingpolypeptides, and secreting proteins. Cells include animal cells used inthe commercial production of biological products, such as insect cells(e.g., Schneider cells, Sf9 cells, Sf21 cells, Tn-368 cells,BTI-TN-5B1-4 cells; see Jarvis, Methods Enzymol. 463: 191-222, 2009; andPotter et al., Int. Rev. Immunol. 10(2-3): 103-112, 1993) and mammaliancells (e.g., CHO or CHO-K1 cells, COS or COS-7 cells, HEK293 cells, PC12cells, HeLa cells, Hybridoma cells; Trill et al., Curr. Opin.Biotechnol. 6(5): 553-560, 1995; Kipriyanov and Little, Mo. Biotechnol.12(2): 173-201, 1999). In one embodiment, the cell is a CHO-K1 cellcontaining the described UPR pathway polynucleotides. For a descriptionof CHO-K1 cells, see also Kao et al., Proc. Nat'l. Acad. Sci. USA 60:1275-1281, 1968.

As used herein, the term “promoter” means a genetic sequence generallyin cis and located upstream of a protein coding sequence, and whichfacilitates the transcription of the protein coding sequence. Promoterscan be regulated (developmental, tissue specific, or inducible(chemical, temperature)) or constitutively active. In certainembodiments, the polynucleotides that encode proteins are operablylinked to a constitutive promoter. By “operably linked”, what is meantis that the protein-encoding polynucleotide is located three-prime(downstream) and cis of the promoter, and under control of the promoter.In certain embodiments, the promoter is a constitutive mammalianpromoter, such as the ubiquitin C promoter (see Schorpp et al., Nucl.Acids Res. 24(9): 1787-1788, 1996); Byun et al., Biochem. Biophys. Res.Comm 332(2): 518-523, 2005) or the CMV-IE promoter (see Addison et al.,J. Gen. Virol. 78(7): 1653-1661, 1997; Hunninghake et al., J. Virol.63(7): 3026-3033, 1989), or the hCMV-IE promoter (human cytomegalovirusimmediate early gene promoter) (see Stinski & Roehr, J. Virol. 55(2):431-441, 1985; Hunninghake et al., J. Virol. 63(7): 3026-3033, 1989).

As used herein, the phrase “integrated cell density”, or “ICD” means thedensity of cells in a culture medium taken as an integral over a periodof time, expressed as cell-days per mL. In some embodiments, the ICD ismeasured around the twelfth day of cells in culture.

As used herein, the term “culture” means both (1) the compositioncomprising cells, medium, and secreted multi-subunit protein, and (2)the act of incubating the cells in medium, regardless of whether thecells are actively dividing or not. Cells can be cultured in a vessel assmall as a 25 mL flask or smaller, and as large as a commercialbioreactor of 10,000 liters or larger. “Medium” refers to the culturemedium, which comprises inter alia nutrients, lipids, amino acids,nucleic acids, buffers and trace elements to allow the growth,proliferation, or maintenance of cells, and the production of themulti-subunit protein by the cells. Cell culture media includeserum-free and hydrolysate-free defined media as well as mediasupplemented with sera (e.g., fetal bovine serum (FBS)) or proteinhydrolysates. Non-limiting examples of media, which can be commerciallyacquired, include RPMI medium 1640, Dulbecco's Modified Eagle Medium(DMEM), DMEM/F12 mixture, F10 nutrient mixture, Ham's F12 nutrientmixture, and minimum essential media (MEM).

As used herein, the phrase “conservatively substituted variant”, asapplied to polypeptides, means a polypeptide having an amino acidsequence with one of more conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent or degree of similarity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, which is herein incorporated by reference. Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartate andglutamate, and 7) sulfur-containing side chains: cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443-45, herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

EMBODIMENTS

The Cell

In one aspect, the invention provides a cell useful in the production ofa protein having therapeutic or research utility. In some embodiments,the protein consists of multiple subunits, which must be properly foldedand assembled to produce sufficient quantities of active protein.Antibodies are an example of multi-subunit proteins having therapeuticor research utility. In some embodiments, the cell harbors a recombinantgenetic construct (i.e., a polynucleotide) that encodes one or more ofthe individual subunits of the multi-subunit protein. In otherembodiments, the genetic construct encoding the individual polypeptidesubunits is naturally occurring, such as for example the nucleic acidsequences encoding the subunits of an antibody in a B cell.

To facilitate the proper assembly and secretion of the multi-subunitprotein, the cell contains a recombinant polynucleotide that encodes astress-induced mannose-binding lectin, which in some embodiments is acomponent of the ERAD. In some embodiments, the stress-inducedmannose-binding lectin is an endoplasmic reticulum degradation-enhancingalpha-mannosidase-like protein 2 (EDEM2). It is envisioned that anyencoded EDEM2 or conservatively-substituted variant can be successfullyemployed in the instant invention. Table 1 lists some examples ofvertebrate EDEM2 proteins. A multiple pairwise comparison of thoseprotein sequences, which was performed using the Clustal W program ofThompson et al., Nucl. Acids Rev. 22(22): 4673-80, 1994 (see also Yuanet al., Bioinformatics 15(10): 862-3, 1999), revealed that each of thedisclosed EDEM2 polynucleotide sequences is at least 69% identical toeach other EDEM2 sequence. A Clustal W comparison of the disclosedmammalian EDEM2 sequences revealed that each sequence is at least 92%identical to the other. Thus, in some embodiments, the cell contains apolynucleotide that encodes an EDEM2 polypeptide having a sequence thatis at least 92% to any one of a mammalian EDEM2. A consensus EDEM2 aminoacid sequence was built by aligning a mouse, rat, hamster, chimpanzee,and human EDEM2 polypeptide amino acid sequences. That consensussequence is depicted as SEQ ID NO: 8. Thus, in some embodiments, thecell contains a polynucleotide that encodes an EDEM2 polypeptide havingan amino acid sequence of SEQ ID NO: 8.

In various embodiments, the cell contains a recombinant polynucleotidethat encodes an EDEM2 polypeptide having an amino acid sequence that isat least 92% identical to the mouse EDEM2 (mEDEM2) amino acid sequence;and in a particular embodiment, the polypeptide is mEDEM2 or aconservatively substituted variant thereof.

In some embodiments, the multi-subunit protein is an antibody, and thecell contains a polynucleotide encoding any one or more of a polypeptidecomprising an amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQID NO: 45, and SEQ ID NO: 46. SEQ ID NO: 43 and 44 each representconsensus sequences of the roughly N-terminal and C-terminal portions,respectively, of particular antibody heavy chains. Thus, thepolynucleotide encoding a protein subunit in one embodiment encodes apolypeptide comprising both SEQ ID NO: 43 and SEQ ID NO: 44. SEQ ID NO:45 and 46 each represent consensus sequences of the roughly N-terminaland C-terminal portions, respectively, of particular antibody lightchains. Thus, the polynucleotide encoding a protein subunit in oneembodiment encodes a polypeptide comprising both SEQ ID NO: 45 and SEQID NO: 46. In some embodiments, in addition to the recombinantpolynucleotide encoding the EDEM2 protein, the cell contains at leasttwo polynucleotides, each of which encodes a particular subunit of themulti-subunit protein. For example, and as exemplified below, the cellcontains a polynucleotide encoding an antibody heavy chain comprising anamino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44, and anotherpolynucleotide encoding an antibody light chain comprising an amino acidsequence of SEQ ID NO: 45 and SEQ ID NO: 46.

In some embodiments, the cell, in addition to containing thestress-response polynucleotide and one or more polynucleotides encodinga polypeptide subunit, as described above, also contains apolynucleotide that encodes an unfolded protein response transcriptionfactor that operates upstream of EDEM2. The upstream transcriptionfactor is in some cases the spliced form of an XBP1. It is envisionedthat any encoded XBP1 can be successfully employed in the instantinvention. Table 2 lists some examples of sequences of vertebrate XBP1spliced-form polypeptides. A multiple pairwise comparison of thosepolypeptide sequences, which was performed using Clustal W (Thompson1994; Yuan 1999), revealed that each of the disclosed spliced XBP1polynucleotide sequences is at least 48% identical to each other XBP1sequence. A Clustal W comparison of the disclosed mammalian XBP1sequences revealed that each sequence is at least 86% identical to theother. Thus, in some embodiments, the cell contains a polynucleotidethat encodes a spliced-form of an XBP1 polypeptide having a sequencethat is at least 86% to any one of a mammalian spliced XBP1. A consensusXBP1 amino acid sequence was built by aligning a mouse, hamster, andhuman XBP1 amino acid sequences. That consensus sequence is depicted asSEQ ID NO: 13. Thus, in some embodiments, the cell contains apolynucleotide that encodes an XBP1 polypeptide having an amino acidsequence of SEQ ID NO: 13.

In various embodiments, the cell contains a polynucleotide that encodesan XBP1 polypeptide having an amino acid sequence that is at least 86%identical to the mouse XBP1 (mXBP1) amino acid sequence (SEQ ID NO: 9);and in a particular embodiment, the polypeptide is mXBP1, or aconservatively substituted variant thereof.

The invention envisions that any cell may be used to harbor thelectin-encoding polypeptide for the production of a properly folded andactive multi-subunit protein. Such cells include the well-known proteinproduction cells such as the bacterium Escherichia coli and similarprokaryotic cells, the yeasts Pichia pastoris and other Pichia andnon-pichia yeasts, plant cell explants, such as those of Nicotiana,insect cells, such as Schneider 2 cells, Sf9 and Sf21, and theTrichoplusia ni-derived High Five cells, and the mammalian cellstypically used in bioproduction, such as CHO, CHO-K1, COS, HeLa, HEK293,Jurkat, and PC12 cells. In some embodiments, the cell is a CHO-K1 or amodified CHO-K1 cell such as that which is taught in U.S. Pat. Nos.7,435,553, 7,514,545, and 7,771,997, as well as U.S. Published PatentApplication No. US 2010-0304436 A1, each of which is incorporated hereinby reference in its entirety.

In some particular embodiments, the invention provides ex vivo a CHO-K1cell that contains (1) a mEDEM2-encoding polynucleotide comprising anucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence that encodes a polypeptide comprising the amino acid sequencesof SEQ ID NO: 43 and 44, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence that encodes apolypeptide comprising the amino acid sequences of SEQ ID NO: 45 and 46.

In one particular embodiment, the invention provides ex vivo a CHO-K1cell that contains (1) a mEDEM2-encoding polynucleotide comprising anucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO:18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence of SEQ ID NO: 23, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 25.

In another particular embodiment, the invention provides ex vivo aCHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotidecomprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence of SEQ ID NO: 31, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 33.

In yet another particular embodiment, the invention provides ex vivo aCHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotidecomprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence of SEQ ID NO: 39, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 41.

The Cell Line

In another aspect, the invention provides a cell line, which comprises aplurality of cells descended by clonal expansion from a cell describedabove. At least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, at least 99%, or about 100% of theconstituent cells of the cell line contain a recombinant polynucleotidethat encodes a stress-induced mannose-binding lectin, which in someembodiments is a component of the ERAD. In some embodiments, thestress-induced mannose-binding lectin is an endoplasmic reticulumdegradation-enhancing alpha-mannosidase-like protein 2 (EDEM2). It isenvisioned that any encoded EDEM2 or conservatively-substituted variantthereof can be successfully employed in the instant invention. Table 1,as discussed in the previous section, lists some examples of vertebrateEDEM2 proteins. In some embodiments, the constituent cell contains apolynucleotide that encodes an EDEM2 polypeptide having a sequence thatis at least 92% identical to any mammalian EDEM2. In some embodiments,the constituent cell contains a polynucleotide that encodes an EDEM2polypeptide having the mammalian consensus amino acid sequence of SEQ IDNO: 8. In some embodiments, the constituent cell contains a recombinantpolynucleotide of SEQ ID NO: 1 or a conservatively substituted variantthereof.

In some embodiments, the multi-subunit protein that is produced by thecell line is an antibody, and the constituent cell of the cell linecontains a polynucleotide encoding any one or more of a polypeptidecomprising an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 44(which represent consensus sequences of the N-terminal and C-terminalportions, respectively, of particular antibody heavy chains), and SEQ IDNO: 45 and SEQ ID NO: 46 (which represent consensus sequences of theN-terminal and C-terminal portions, respectively, of particular antibodylight chains). In some embodiments, in addition to the recombinantpolynucleotide encoding the EDEM2 protein, the constituent cell of thecell line contains at least two polynucleotides, each of which encodes aparticular subunit of the multi-subunit protein. For example, theconstituent cell contains a polynucleotide encoding an antibody heavychain comprising an amino acid sequence of SEQ ID NO: 43 and SEQ ID NO:44, and another polynucleotide encoding an antibody light chaincomprising an amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 46.

In some embodiments, the constituent cell, in addition to containing thestress-response polynucleotide and one or more polynucleotides encodinga polypeptide subunit, as described above, also contains apolynucleotide that encodes an unfolded protein response transcriptionfactor, which operates upstream of EDEM2, such as a spliced form of anXBP1. It is envisioned that any encoded XBP1 can be successfullyemployed in the instant invention. Table 2, as discussed in thepreceding section, lists some examples of sequences of vertebrate XBP1spliced-form polypeptides. Clustal W analysis of those sequencesrevealed that each of the disclosed spliced XBP1 polynucleotidesequences is at least 48% identical to each other XBP1 sequence; and acomparison of the mammalian XBP1 sequences revealed that each sequenceis at least 86% identical to the other. Thus, in some embodiments, theconstituent cell of the cell line contains a polynucleotide that encodesa spliced-form of an XBP1 polypeptide having a sequence that is at least86% to any one of a mammalian spliced XBP1. In some embodiments, theconstituent cell contains a polynucleotide that encodes an XBP1polypeptide having a consensus amino acid sequence of SEQ ID NO: 13.

In various embodiments, the cell contains a polynucleotide that encodesan XBP1 polypeptide having an amino acid sequence that is at least 86%identical to the mouse XBP1 (mXBP1) amino acid sequence (SEQ ID NO: 9);and in a particular embodiment, the polypeptide is mXBP1 of SEQ ID NO:9, or a conservatively substituted variant thereof.

The invention envisions that the cell line comprises constituent cellswhose parent is selected from a list of well-known protein productioncells such as, e.g., the bacterium Escherichia coli and similarprokaryotic cells, the yeasts Pichia pastoris and other Pichia andnon-pichia yeasts, plant cell explants, such as those of Nicotiana,insect cells, such as Schneider 2 cells, Sf9 and Sf21, and theTrichoplusia ni-derived High Five cells, and the mammalian cellstypically used in bioproduction, such as CHO, CHO-K1, COS, HeLa, HEK293,Jurkat, and PC12 cells. In some embodiments, the cell is a CHO-K1 or amodified CHO-K1 cell, such as that which is taught in U.S. Pat. Nos.7,435,553, 7,514,545, and 7,771,997, as well as U.S. Published PatentApplication No. US 2010-0304436 A1.

In some embodiments, the cell line, which is cultured in media, iscapable of producing the multi-subunit protein and secreting theproperly assembled multi-subunit protein into the media to a titer thatis at least 3 g/L, at least 5 g/L, or at least 8 g/L.

Furthermore, the constituent cells of the cell line are capableproliferating in culture to such an extent as to attain an integratedcell density that is about 30% greater than the integrated cell densityof a cell line that does not contain the recombinant polynucleotideencoding the stress-induced mannose-binding lectin. In some cases, thecell line is able to attain an integrated cell density that is at leastabout 50% greater, at least 60% greater, or at least 90% greater thanthe integrated cell density of a cell line that does not contain therecombinant polynucleotide that encodes a stress-induced mannose-bindinglectin. In some embodiments, the integrated cell density of the cellline is assessed after about 12 days in culture.

In some particular embodiments, the invention provides a cell-linecomprising clonally-derived constituent cells, wherein the constituentcell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotidecomprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence that encodes a polypeptide comprising the amino acid sequencesof SEQ ID NO: 43 and 44, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence that encodes apolypeptide comprising the amino acid sequences of SEQ ID NO: 45 and 46.

In one particular embodiment, the invention provides a cell-linecomprising clonally-derived constituent cells, wherein the constituentcell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotidecomprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence of SEQ ID NO: 23, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 25.

In another particular embodiment, the invention provides a cell-linecomprising clonally-derived constituent cells, wherein the constituentcell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotidecomprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence of SEQ ID NO: 31, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 33.

In yet another particular embodiment, the invention provides a cell-linecomprising clonally-derived constituent cells, wherein the constituentcell is a CHO-K1 cell that contains (1) a mEDEM2-encoding polynucleotidecomprising a nucleotide sequence of SEQ ID NO: 16, (2) an XBP1-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 18, (3) anantibody heavy chain-encoding polynucleotide comprising a nucleotidesequence of SEQ ID NO: 39, and (4) an antibody light chain-encodingpolynucleotide comprising a nucleotide sequence of SEQ ID NO: 41.

The EDEM2 Polynucleotide

In another aspect, the invention provides a polynucleotide that encodesan EDEM2 protein. The EDEM2-encoding polynucleotide is recombinant andcan be manufactured, stored, used or expressed in vitro, as in a testtube, or an in vitro translation system, or in vivo, such as in a cell,which can be ex vivo, as in a cell culture, or in vivo, as in anorganism. In some embodiments, the EDEM2-encoding polynucleotide iswithin a gene, meaning that it is under the control of and down streamof a promoter, and upstream of a polyadenylation site. TheEDEM2-encoding polynucleotide or gene can be within a plasmid or othercircular or linear vector. The EDEM2-encoding polynucleotide or gene canbe within a circular or linear DNA construct, which can be within a cellas an episome or integrated into the cellular genome.

As described above, the EDEM2-encoding polynucleotide encodes anyortholog, homolog or conservatively substituted EDEM2 polypeptide ofTable 1, or an EDEM2 polypeptide having an amino acid sequence that isat least 92% identical to any one of SEQ ID NO: 1-5 and 8, including themammalian consensus sequence of SEQ ID NO: 8.

In some cases, the recombinant or isolated EDEM2-encoding polynucleotideis operably linked to a mammalian promoter. The promoter can be anypromoter, but in some cases it is a mammalian promoter, such as forexample a ubiquitin C promoter.

In a particular embodiment, the EDEM2-encoding polynucleotideessentially consists of, from 5′ to 3′, a promoter, such as a ubiquitinC promoter, followed by an optional intron, such as a beta globinintron, followed by an EDEM2 coding sequence, followed by apolyadenylation sequence, such as an SV40 pA sequence. A specificexample, which is also a particular embodiment, of such anEDEM2-encoding polynucleotide is described by SEQ ID NO: 16. Conservedvariants of that sequence are also envisioned to be embodiments of theinvention.

In some cases, the recombinant EDEM2-encoding polynucleotide is part ofa plasmid, which can be linear, circular, episomal, integrated, a staticDNA construct, or a vector for delivering the EDEM2 gene or expressingthe EDEM2 protein. In one particular embodiment, the plasmid contains(1) an EDEM2 gene, which is under the control of a ubiquitin C promoterand terminates with an SV40 polyadenylation signal, and (2) a selectablemarker, such as a polynucleotide encoding a polypeptide that confersresistance to zeocin or a polynucleotide encoding a polypeptide thatconfers resistance to neomycin, under the control of a promoter, such asan SV40 promoter, and terminated with a polyadenylation sequence, suchas a PGK pA sequence. In one particular embodiment, the plasmidcomprises, in a circular format running in a 5′ to 3′ direction, aubiquitin C promoter, a beta globin intron, an EDEM2 coding sequence, anSV40 pA sequence, an SV40 promoter, a neomycin-resistance codingsequence, and a PGK pA sequence. A specific example of this embodimentis exemplified by a plasmid having the sequence of SEQ ID NO: 14. Inanother particular embodiment, the plasmid comprises, in a circularformat running in a 5′ to 3′ direction, a ubiquitin C promoter, a betaglobin intron, an EDEM2 coding sequence, an SV40 pA sequence, an SV40promoter, a zeocin-resistance coding sequence, and a PGK pA sequence. Aspecific example of this embodiment is exemplified by a plasmid havingthe sequence of SEQ ID NO: 15.

The XBP1 Polynucleotide

In another aspect, the invention provides a polynucleotide that encodesan XBP1 protein. The XBP1-encoding polynucleotide is recombinant and canbe manufactured, stored, used or expressed in vitro, as in a test tube,or an in vitro translation system, or in vivo, such as in a cell, whichcan be ex vivo, as in a cell culture, or in vivo, as in an organism. Insome embodiments, the XBP1-encoding polynucleotide is within a gene,meaning that it is under the control of and down stream of a promoter,and upstream of a polyadenylation site. The XBP1-encoding polynucleotidecan be within a plasmid or other circular or linear vector. TheXBP1-encoding polynucleotide or gene can be within a circular or linearDNA construct, which can be within a cell as an episome, or integratedinto the cellular genome.

As described above, the XBP1-encoding polynucleotide encodes anyortholog, homolog or conservatively substituted XBP1 polypeptide ofTable 2, or an XBP1 polypeptide having an amino acid sequence that is atleast 86% identical to any one of SEQ ID NO: 9, 10, and 11, includingthe mammalian consensus sequence of SEQ ID NO: 13.

In some cases, the recombinant or isolated XBP1-encoding polynucleotideis operably linked to a mammalian promoter. The promoter can be anypromoter, but in some cases it is a mammalian promoter, such as forexample a ubiquitin C promoter.

In a particular embodiment, the XBP1-encoding polynucleotide essentiallyconsists of, from 5′ to 3′, a promoter, such as a ubiquitin C promoter,followed by an optional intron, such as a beta globin intron, followedby an XBP1 coding sequence, followed by a polyadenylation sequence, suchas an SV40 pA sequence. SEQ ID NO: 18 describes an example of anXBP1-encoding polynucleotide. Conserved variants of that exemplarysequence are also envisioned to be embodiments of the invention.

In some cases, the recombinant XBP1-encoding polynucleotide is part of aplasmid, which can be linear, circular, episomal, integrated, a staticDNA construct, or a vector for delivering the XBP1 gene or expressingthe spliced and active XBP1 protein. In one particular embodiment, theplasmid contains (1) an XBP1 gene, which is under the control of aubiquitin C promoter and terminates with an SV40 polyadenylation signal,and (2) a selectable marker, such as a polynucleotide encoding apolypeptide that confers resistance to zeocin or a polynucleotideencoding a polypeptide that confers resistance to neomycin, under thecontrol of a promoter, such as an SV40 promoter, and terminated with apolyadenylation sequence, such as a PGK pA sequence. In one particularembodiment, the plasmid comprises, in a circular format running in a 5′to 3′ direction, a ubiquitin C promoter, a beta globin intron, an XBP1coding sequence, an SV40 pA sequence, an SV40 promoter, azeocin-resistance coding sequence, and a PGK pA sequence. A specificexample of this embodiment is exemplified by a circular plasmid havingthe sequence of SEQ ID NO: 17.

The Antibody Heavy and Light Chain-Encoding Polynucleotides

In another aspect, the invention provides a polynucleotide that encodesan antibody heavy chain polypeptide (HC). The HC-encoding polynucleotideis recombinant and can be manufactured, stored, used or expressed invitro, as in a test tube, or an in vitro translation system, or in vivo,such as in a cell, which can be ex vivo, as in a cell culture, or invivo, as in an organism. In some embodiments, the HC-encodingpolynucleotide is within a gene, meaning that it is under the control ofand down stream from a promoter, and upstream of a polyadenylation site.The HC-encoding polynucleotide may be within a plasmid or other circularor linear vector. The HC-encoding polynucleotide or gene may be within acircular or linear DNA construct, which may be within a cell as anepisome or integrated into the cellular genome.

In some cases, the recombinant or isolated HC-encoding polynucleotide isoperably linked to a mammalian promoter. The promoter can be anypromoter, but in some cases it is a mammalian promoter, such as forexample a ubiquitin C promoter or an hCMV-IE promoter.

In a particular embodiment, the HC-encoding polynucleotide is an HCgene, which essentially comprises, from 5′ to 3′, a promoter, forexample an hCMV-IE promoter, followed by an optional intron, such as abeta globin intron, followed by a heavy chain coding sequence, such asfor example a sequence encoding an amino acid sequence of SEQ ID NO: 43and 44, SEQ ID NO: 19, SEQ ID NO: 27, or SEQ ID NO: 35, followed by apolyadenylation sequence, for example an SV40 pA sequence. A specificexample of an HC gene is described by SEQ ID NO: 23, SEQ ID NO: 31, orSEQ ID NO: 39. Conserved variants of any one of these sequences are alsoenvisioned to be embodiments of the invention.

In some cases, the recombinant HC-encoding polynucleotide is part of aplasmid, which can be linear, circular, episomal, integrated, a staticDNA construct, or a vector for delivering the heavy chain gene orexpressing the heavy chain sububunit. In one particular embodiment, theplasmid contains (1) an HC gene, which is under the control of anhCMV-IE promoter and terminates with an SV40 polyadenylation signal, and(2) a selectable marker, such as a polynucleotide encoding a polypeptidethat confers resistance to hygromycin, under the control of a promoter,such as an SV40 promoter, and terminated with a polyadenylationsequence, such as a PGK pA sequence. In one particular embodiment, theplasmid comprises, in a circular format running in a 5′ to 3′ direction,an hCMV-IE promoter, a beta globin intron, an antibody heavy chaincoding sequence (which encodes a HC having an amino acid of SEQ ID NO:43 and 44, SEQ ID NO: 19, SEQ ID NO: 27, or SEQ ID NO: 35), an SV40 pAsequence, an SV40 promoter, a hygromycin-resistance coding sequence, anda PGK pA sequence. A specific example and particular embodiment of sucha plasmid containing an HC gene is described by SEQ ID NO: 24, SEQ IDNO: 32, or SEQ ID NO: 40. Conserved variants of any one of thesesequences are also envisioned to be embodiments of the invention.

In another aspect, the invention provides a polynucleotide that encodesan antibody light chain polypeptide (LC). The LC-encoding polynucleotideis recombinant and can be manufactured, stored, used or expressed invitro, as in a test tube, or an in vitro translation system, or in vivo,such as in a cell, which can be ex vivo, as in a cell culture, or invivo, as in an organism. In some embodiments, the LC-encodingpolynucleotide is within a gene, meaning that it is under the control ofand down stream from a promoter, and upstream of a polyadenylation site.The LC-encoding polynucleotide or gene may be within a plasmid or othercircular or linear vector. The LC-encoding polynucleotide or gene may bewithin a circular or linear DNA construct, which may be within a cell asan episome or integrated into the cellular genome.

In some cases, the recombinant or isolated LC-encoding polynucleotide isoperably linked to a mammalian promoter. The promoter can be anypromoter, but in some cases it is a mammalian promoter, such as, e.g., aubiquitin C promoter or an hCMV-IE promoter.

In a particular embodiment, the LC-encoding polynucleotide is an LCgene, which essentially comprises, from 5′ to 3′, a promoter, forexample an hCMV-IE promoter, followed by an optional intron, such as abeta globin intron, followed by a light chain coding sequence, such asfor example a sequence encoding an amino acid sequence of SEQ ID NO: 45and 46, SEQ ID NO: 21, SEQ ID NO: 29, or SEQ ID NO: 37, followed by apolyadenylation sequence, such as an SV40 pA sequence. A specificexample and particular embodiment of such an LC gene is described by SEQID NO: 25, SEQ ID NO: 33, or SEQ ID NO: 41. Conserved variants of anyone of these sequences are also envisioned to be embodiments of theinvention.

In some cases, the recombinant LC-encoding polynucleotide is part of aplasmid, which may be linear, circular, episomal, integrated, a staticDNA construct, or a vector for delivering the light chain gene orexpressing the light chain sububunit. In one particular embodiment, theplasmid contains (1) an LC gene, which is under the control of anhCMV-IE promoter and terminates with an SV40 polyadenylation signal, and(2) a selectable marker, such as a polynucleotide encoding a polypeptidethat confers resistance to hygromycin, under the control of a promoter,such as an SV40 promoter, and terminated with a polyadenylationsequence, such as a PGK pA sequence. In one particular embodiment, theplasmid comprises, in a circular format running in a 5′ to 3′ direction,an hCMV-IE promoter, a beta globin intron, an antibody light chaincoding sequence (which encodes a LC having an amino acid of SEQ ID NO:45 and 46, SEQ ID NO: 21, SEQ ID NO: 29, or SEQ ID NO: 37), an SV40 pAsequence, an SV40 promoter, a hygromycin-resistance coding sequence, anda PGK pA sequence. A specific example and particular embodiment of sucha plasmid containing an LC gene is described by SEQ ID NO: 26, SEQ IDNO: 34, or SEQ ID NO: 42. Conserved variants of any one of thesesequences are also envisioned to be embodiments of the invention.

Methods of Manufacturing Multi-Subunit Proteins

In another aspect, the invention provides a method for manufacturing amulti-subunit protein by culturing a cell, or a constituent cell of acell line, which is capable of producing and secreting relatively largeamounts of a properly assembled multi-subunit protein, in a medium,wherein the multi-subunit component is secreted into the medium at arelatively high titer. The cell utilized in this manufacturing processis a cell described in the foregoing aspects, which contains an ERADlectin-encoding polynucleotide described herein.

Methods of culturing cells, and in particular mammalian cells, for thepurpose of producing useful recombinant proteins is well-known in theart (e.g., see De Jesus & Wurm, Eur. J. Pharm. Biopharm. 78:184-188,2011, and references cited therein). Briefly, cells containing thedescribed polynucleotides are cultured in media, which may contain seraor hydrolysates, or may be chemically defined and optimized for proteinproduction. The cultures may be fed-batch cultures or continuouscultures, as in a chemostat. The cells may be cultured in lab bench sizeflasks (.about.25 mL), production scale-up bioreactors (1-5 L), orindustrial scale bioreactors (5,000-25,000 L). Production runs may lastfor several weeks to a month, during which time the multi-subunitprotein is secreted into the media.

The subject cell has an enhanced ability to produce and secrete properlyassembled multi-subunit proteins. In some embodiments, the multi-subunitprotein, for example an antibody, is secreted into the media at a rateof at least 94 pg/cell/day, at least 37 pg/cell/day, or at least 39pg/cell/day. In some embodiments, the multi-subunit protein attains atiter of at least at least 3 g/L, at least 5 g/L, at least 6 g/L, or atleast 8 g/L after about twelve days of culture.

Furthermore, the subject cell has an enhanced ability to proliferate andattain a relatively high cell density, further optimizing productivity.In some embodiments, the cell or cell-line seed train attains anintegrated cell density in culture of at least 5×10⁷ cell-day/mL, atleast 1×10⁸ cell-day/mL or at least 1.5×10⁸ cell-day/mL.

Optionally, the secreted multi-subunit protein is subsequently purifiedfrom the medium into which it was secreted. Protein purification methodsare well-known in the art (see e.g., Kelley, mAbs 1(5):443-452). In someembodiments, the protein is harvested by centrifugation to remove thecells from the liquid media supernatant, followed by variouschromatography steps and a filtration step to remove inter alia virusesand other contaminants or adulterants. In some embodiments, thechromatography steps include an ion exchange step, such ascation-exchange or anion-exchange. Various affinity chromatographicmedia may also be employed, such as protein A chromatography for thepurification of antibodies.

Optionally, the manufacturing method may include the antecedent steps ofcreating the cell. Thus, in some embodiments, the method ofmanufacturing the multi-subunit protein comprises the step oftransfecting the cell with the vector that encodes the stress-inducedmannose-binding lectin, as described above, followed by selecting stableintegrants thereof. Non-limiting examples of vectors include thosegenetic constructs that contain a polynucleotide that encodes an EDEM2having an amino acid sequence of any one of SEQ ID NO: 1-8, an aminoacid sequence that is at least 92% identical to any one of SEQ ID NO:1-8, or any one of a conservatively substituted variant of SEQ ID NO:1-8. Useful vectors also include, for example, a plasmid harboring thegene of SEQ ID NO: 16, the plasmid of SEQ ID NO: 15, and the plasmid ofSEQ ID NO: 14. One should keep in mind that the plasmid sequences (e.g.,SEQ ID NO: 14, 15, 17, 24, 26, 32, 34, 40, and 42) are circularsequences described in a linear manner in the sequence listing. Thus, inthose cases, the 3-prime-most nucleotide of the written sequence may beconsidered to be immediately 5-prime of the 5-prime-most nucleotide ofthe sequence as written. In the example of the plasmid of SEQ ID NO: 14,transformants are selected through resistance to neomycin; for SEQ IDNO: 15, by selection through ZEOCIN resistance.

Detailed methods for the construction of polynucleotides and vectorscomprising same, are described in U.S. Pat. Nos. 7,435,553 and7,771,997, which are incorporated herein by reference, and in, e.g.,Zwarthoff et al., J. Gen. Virol. 66(4):685-91, 1985; Mory et al., DNA.5(3):181-93, 1986; and Pichler et al., Biotechnol. Bioeng.108(2):386-94, 2011.

The starting cell, into which the vector that encodes the stress-inducedmannose-binding lectin is placed, may already contain the constructs orgenetic elements encoding or regulating the expression of the subunitsof the multi-subunit protein, or XBP1 for those embodiments utilizingXBP1. Alternatively, the vector that encodes the stress-inducedmannose-binding lectin may be put inside the cell first, and followed bythe other constructs.

Multi-Subunit Proteins Manufactured by the Process

In another aspect, the invention provides a multi-subunit protein thatis made according to the process disclosed herein. Given the inclusionof one or more elements that facilitate the proper folding, assembly,and post-translational modification of a multi-subunit protein, such asan antibody, one of ordinary skill in the art would reasonably expectsuch a protein to have distinct structural and functional qualities. Forexample, an antibody manufactured by the disclosed process is reasonablybelieved to have a particular glycosylation pattern and a quantitativelygreater proportion of non-aggregated heterotetramers.

EXAMPLES

The following examples are presented so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by mole, molecular weight is averagemolecular weight, percent concentration (%) means the mass of the solutein grams divided by the volume of the solution in milliliters times 100%(e.g., 10% substance X means 0.1 gram of substance X per milliliter ofsolution), temperature is in degrees Centigrade, and pressure is at ornear atmospheric pressure.

Example 1 Cell Lines

CHO-K1 derived host cell line was transfected with two plasmids encodingheavy and light chain of a human antibody. Both plasmids contain the hphgene conferring resistance to hygromycin B (Asselbergs and Pronk, 1992,Mol. Biol. Rep., 17(1):61-70). Cells were transfected using LIPOFECTAMINreagent (Invitrogen cat.#18324020). Briefly, one day before transfection3.5 million cells were plated on a 10 cm plate in complete F12(Invitrogen cat.#11765) containing 10% fetal bovine serum (FBS)(Invitrogen cat.#10100). On the day of transfection the cells werewashed once and medium was replaced with OPTIMEM from (Invitrogencat.#31985). DNA/Lipofectamin complexes were prepared in OPTIMEM mediumand then added to the cells. The medium was changed again to thecomplete F12 with 10% FBS 6 hours later. The stable integration of theplasmids was selected using hygromycin B selection agent at 400 μg/ml.Clonal antibody expressing cell lines were isolated using the FASTRtechnology (described in the U.S. Pat. No. 6,919,183, which is hereinincorporated by reference for the expression in a cell of a cell surfacecapture molecule that binds to a protein of interest to aid in theselection of those cells with high expression of the protein ofinterest).

The antibody expressing lines were then re-transfected with the EDEM2encoding plasmid. EDEM2 plasmids contained either neomycinphosphotransferase (plasmid construct designated “p3”) or sh ble(plasmid “p7”) genes to confer resistance to either G418 or zeocinrespectively. The same transfection method was used. Depending on theselectable marker, cells were selected with either G418 or zeocin at 400μg/ml or 250 μg/ml, respectively. The clonal cell lines were thenisolated using FASTR technology.

TABLE 3 CELL LINES Name Enhancers Constructs Protein C1 EDEM2 + XBP1HC/LC = p1/p2 anti-Ang2 C2 XBP1 EDEM2 = p3 XBP1 = p4 C3 EDEM2 + XBP1HC/LC = p5/p6 anti-GDF8 C4 XBP1 EDEM2 = p7 C5 EDEM2 XBP1 = p4 C6 EDEM2 +XBP1 HC/LC = p8/p9 anti-AngPtI4 C7 XBP1 EDEM2 = p3 XBP1 = p4

Example 2

The antibody production was evaluated in a scaled-down 12-day fed batchprocess using shaker flasks. In this method the cells were seeded in ashaker flask at the density of 0.8 million cells per mL in theproduction medium (defined media with high amino acid). The culture wasmaintained for about 12 days, and was supplemented with three feeds aswell as glucose. The viable cell density, and antibody titer weremonitored throughout the batch.

To determine the effect of mEDEM2 on enhanced protein production, theproduction of proteins by CHO cell lines containing mEDEM2 and mXBP1were compared to production by control cells that contained mXBP1, butnot mEDEM2. Protein titers were higher in those cell lines expressingmEDEM2 versus those cell lines that did not express mEDEM2.

TABLE 4 TITERS Production rate Titer g/L Cell Line Enhancers(pg/cell/day) (% increase) C1 EDEM2 + XBP1 39 8.1 (93)  C2 XBP1 39 4.2C3 EDEM2 + XBP1 37 5.9 (55)  C8 XBP1 32 3.8 C6 EDEM2 + XBP1 94 5.3 (152)C7 XBP1 52 2.1 C5 EDEM2 29 3.1 (343) C9 — 9 0.7

Example 3 Integrated Cell Days

Integrated Cell Density (“ICD”) is a phrase used to describe the growthof the culture throughout the fed batch process. In the course of the12-day production assay, we monitored viable cell density on days 0, 3,5, 7, 10, and 12. This data was then plotted against time. ICD is theintegral of viable cell density, calculated as the area under the celldensity curve. EDEM2 transfected lines have higher ICD in a 12-day fedbatch process (see Table 5).

TABLE 5 INTEGRATED CELL DENSITIES ICD 10⁶ cell-day/mL Cell LineEnhancers (% increase) C1 EDEM2 + XBP1 205 (93) C2 XBP1 106 C3 EDEM2 +XBP1 157 (34) C4 XBP1 117 C6 EDEM2 + XBP1  56 (51) C7 XBP1  37 C5 EDEM2116 (59) C9 —  73

Example 4 Anti-GDF8 Antibody Production

The effect of ectopic expression of EDEM2, XBP1, or both on theproduction of an anti-GDF8 antibody having a heavy chain sequence of SEQID NO: 19 and a light chain sequence of SEQ ID NO: 21 was examined.Individual cell-lines were examined for titer and integrated celldensity and placed into “bins”, or ranges of values. Ectopic expressionof EDEM2 significantly increased the number of cell lines that expressantibody in the 5-6 g/L titer range. The combination of XBP1 and EDEM2showed more than an additive effect toward the increase in high titercell lines. The expression of EDEM2 in the antibody secreting cells alsosignificantly increased the number of cell lines that attain a high ICD(see Table 6).

TABLE 6 ICD Bins con- Titre Bins (g/L) (10⁶ cell-day/mL) struct <1 1-33-5 5-6 30-50 50-100 100-200 E + 0% 33.3% 44.4%   22.2%   11.1% 50%38.9%   X X 0% 37.5% 54% 8.3%   14.3% 85.7%    0% E 0%   33% 60% 7%   0%27% 73% — 82%   18%  0% 0%   13% 67% 21%

Example 5 Productivity and Stability of EDEM2-Expressing Cells

The effect of ectopic expression of XBP1 or EDEM2 on the production of amonospecific antibody of interest (identified as clonal cell lines RGC91or RGC92, respectively) was examined. Individual cell lines wereexamined for protein titer and integrated cell density, as well asstability.

Modified CHO K1 host cells stably expressing XBP1 (RGC91) or EDEM2(RGC92) at a transcriptionally active locus (U.S. Pat. No. 8,389,239B2,issued Mar. 5, 2013) were transfected with a recombinant plasmid vectorcomprising the antibody gene of interest and a hygromycin resistancegene (hyg).

400 μg/mL hygromycin was used for selection of transfected cells.Positive integrants expressing the antibody of interest (randomlyintegrated in the CHO genome), and also stably expressing either XBP1 orEDEM2, were confirmed and isolated by fluorescence-activated cellsorting (FACS) analysis. The isolated clones were expanded in suspensioncultures in serum-free production medium. Clones were isolated fromselected pools and were subjected to a 12 day fed batch productivityassay, and the protein titer of the antibody of interest was determined.Integrated cell density is calculated by measuring viable cell count ona given day in the production assay (counts are taken every 3 days andplotted on a curve against cell count).

As shown in FIG. 1A, the average protein titers for clones isolated fromRGC91 and RGC92 was 4.2 and 5.2, respectively (for 24 representativeantibody-expressing clones). Ectopic expression of EDEM2 increased thenumber of clones that attain antibody titer above 5 g/L (FIG. 1A)compared to XBP1-expressing clones. Clonal cell lines expressing EDEM2isolated from the RGC92 host also maintained higher (25%-100% higher)integrated cell densities when compared to clones isolated fromXBP1-expressing RGC91 host (see FIG. 1B). EDEM2 clones established anICD greater than 100 (FIG. 1B) in most clones tested. Clones isolatedfrom EDEM2 expressing RGC92 host also resulted in significantly higherstability (FIG. 2B), as observed in flow cytometry-based autologoussecretion trap (FASTR) scans (for reference, U.S. Pat. No. 6,919,183B2,issued Jul. 19, 2005) showing a homogenous producing population, in therepresentative sample of 24 clonal cell lines tested. Many of the clonesisolated from XBP1 expressing RGC91 host appear to have a non-producingheterogeneous cell population (FIG. 2A). Without being bound to any onetheory, EDEM2 facilitated the removal of misfolded proteins in the highexpressing clones, thereby reducing stress in the cell during proteinproduction and resulting in a more stable cell population.

What is claimed is:
 1. A eukaryotic cell comprising: a. a firstpolynucleotide comprising a nucleic acid sequence encoding a firstproduction enhancer protein comprising an amino acid sequence that is atleast 92% identical to SEQ ID NO:9; and b. a second polynucleotidecomprising a nucleic acid encoding a multi-subunit protein comprising anamino acid sequence of SEQ ID NO:44, wherein the cell produces themulti-subunit protein at a rate of at least 32 pg/cell/day.
 2. The cellof claim 1 wherein the first polynucleotide further comprises aconstitutive promoter operably linked to the nucleic acid encoding thefirst production enhancer protein.
 3. The cell of claim 2 wherein theconstitutive promoter of the first polynucleotide is selected from thegroup consisting of ubiquitin C promoter, CMV-IE promoter, and SV40promoter.
 4. The cell of claim 1 wherein the first polynucleotide isintegrated at a transcriptionally active locus of the cell.
 5. The cellof claim 1 wherein the first production enhancer protein comprises theamino acid sequence selected from the group consisting of SEQ ID NO: 13,SEQ ID NO:9, and SEQ ID NO:10.
 6. The cell of claim 1 wherein the cellfurther comprises a third polynucleotide comprising a nucleic acidsequence encoding a second production enhancer protein comprising anamino acid sequence that is at least 92% identical to SEQ ID NO:
 1. 7.The cell of claim 6 wherein the third polynucleotide further comprises aconstitutive promoter operably linked to the nucleic acid encoding thesecond production enhancer protein.
 8. The cell of claim 7 wherein theconstitutive promoter is selected from the group consisting of ubiquitinC promoter, CMV-IE promoter, and SV40 promoter.
 9. The cell of claim 6wherein the second production enhancer protein comprises the amino acidsequence selected from the group consisting of SEQ ID NO: 8, SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6.
 10. The cell of claim 6 wherein the cell produces themulti-subunit protein at a rate of at least 37 pg/cell/day.
 11. The cellof claim 1 wherein the multi-subunit protein is an antibody.
 12. Thecell of claim 11 wherein the antibody comprises a light chain with anamino acid sequence of SEQ ID NO:46.
 13. The cell of claim 12 whereinthe antibody is an anti-GDF8 antibody, an anti-Ang2 antibody, or ananti-AngPtl4 antibody.
 14. The cell of claim 1 wherein the cell is amodified CHO-K1 cell.
 15. The cell of claim 6 further comprising afourth polynucleotide encoding a cell surface capture molecule thatbinds the multi-subunit protein.
 16. A cell line comprising a pluralityof cells descended by clonal expansion from the cell of claim
 1. 17. Amethod of making a multi-subunit protein comprising: a. culturing inmedia a plurality of eukaryotic cells descended by clonal expansion froma eukaryotic cell comprising: i. a first polynucleotide comprising anucleic acid encoding a first production enhancer protein comprising anamino acid sequence that is at least 92% identical to SEQ ID NO:9, andii. a second polynucleotide comprising a nucleic acid encoding amulti-subunit protein comprising an amino acid sequence of SEQ ID NO:44;b. allowing the cells to secrete the multi-subunit protein into themedia at a rate greater than 32 pg/cell/day; and c. purifying themulti-subunit protein from the media by affinity chromatography, ionexchange chromatography, or a combination of affinity chromatography andion exchange chromatography.
 18. The method of claim 17 wherein thefirst production enhancer protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQID NO:13.
 19. The method of claim 17 wherein the nucleic acid encodingthe first production enhancer protein is operably linked to aconstitutive ubiquitous promoter.
 20. The method of claim 19 wherein theconstitutive promoter is selected from the group consisting of ubiquitinC promoter, CMV-IE promoter, and SV40 promoter.
 21. The method of claim18 wherein the first polynucleotide comprises the nucleic acid sequenceof SEQ ID NO:18.
 22. The method of claim 21 wherein the firstpolynucleotide comprises the nucleic acid sequence of SEQ ID NO:17. 23.The method of claim 17 further comprising prior to step a. the steps of:aa. in any order, i. transfecting the cell with the firstpolynucleotide, and ii. transfecting the cell with the secondpolynucleotide; and bb. clonally expanding the cell comprising the firstpolynucleotide and the second polynucleotide.
 24. The method of claim 17wherein the cell further comprises a third polynucleotide comprising anucleic acid encoding a second production enhancer protein comprising anamino acid sequence that is at least 92% identical to SEQ ID NO:1, andallowing the cells at step (b) to secrete the multi-subunit protein intothe media at a rate ≥37 pg/cell/day.
 25. The method of claim 24 whereinthe second production enhancer protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:8.
 26. Themethod of claim 24 wherein the nucleic acid encoding the secondproduction enhancer protein is operably linked to a constitutivepromoter.
 27. The method of claim 26 wherein the constitutive promoteris selected from the group consisting of ubiquitin C promoter, CMV-IEpromoter, and SV40 promoter.
 28. The method of claim 25 wherein thethird polynucleotide comprises the nucleic acid sequence of SEQ IDNO:16.
 29. The method of claim 28 wherein the third polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:14 or
 15. 30. Themethod of claim 24 further comprising prior to step a. the steps of: aa.in any order, i. transfecting the cell with the first polynucleotide,ii. transfecting the cell with the second polynucleotide, and iii.transfecting the cell with the third polynucleotide; and bb. clonallyexpanding the cell comprising the first polynucleotide, the secondpolynucleotide, and the third polynucleotide.
 31. The method of claim 17wherein the multi-subunit protein accumulates in the media to a titer of≥2.1 g/L, ≥3.8 g/L, or ≥4.2 g/L.
 32. The method of claim 24 wherein themulti-subunit protein accumulates in the media to a titer of ≥5.3 g/L,≥5.9 g/L, or ≥8.1 g/L.
 33. The method of claim 17 wherein themulti-subunit protein is an antibody.
 34. The method of claim 33 whereinthe antibody further comprises a polypeptide with an amino acid sequenceof SEQ ID NO:46.
 35. The method of claim 34 wherein the antibody is ananti-GDF8 antibody, an anti-Ang2 antibody, or an anti-AngPtl4 antibody.